Hepatitis A virus (HAV) is a picornavirus with a single-strand positive sense RNA genome of approximately 7500 nucleotides. The wild-type strain of HAV grows poorly in cell culture, generally is not cytopathic, and virus yields are low. Cell culture-adapted mutants generally grow significantly more efficiently in cell culture and are attenuated for marmosets and chimpanzees. The objectives of this project are to determine the genetic basis for virulence and adaptation to cell culture of HAV in order to develop a strain of HAV suitable for use as an attenuated vaccine. The following advances in our understanding of HAV were made. In an effort to increase replicative capacity, chimeric viruses were constructed from two or more HAV strains, including a virulent human strain, an attenuated strain, a vaccine strain, a cytopathogenic strain and a simian strain. A small number of mutations in the 2B and 2C genes of the cytopathogenic strain conferred the large focus phenotype but not the lytic phenotype of the cytopathogenic virus. HAV/7 chimeras containing the 2C gene of the simian virus grew less efficiently than did HAV/7 but intragenic 2C chimeras grew at an intermediate level. Chimeric viruses were constructed and assayed for the purpose of defining virulence genes. We identified the 2A gene as a second major HAV/7 determinant of attenuation in tamarins and obtained evidence that mutations in the 2A and 2C genes are almost totally responsible for the attenuation of the virus. The genetic determinants of virulence could be conferred through the 2C gene of a simian virus. The 5' noncoding mutations required for growth in MRC-5 cells resulted in lower peak serum liver enzyme levels in combination with significant histopathology and high levels of virus excretion. A full length infectious cDNA of the attenuated HAV MRC-5 cell-adapted vaccine strain was constructed to serve as a genetic repository for the vaccine strain and to permit detailed molecular analysis of the virus it encodes. An ELISA based on recombinant 3C proteinase was developed to distinguish an antibody response due to HAV infection from one due to immunization with an inactivated vaccine.